Epithelial Ovarian Cancer Cell Migration Research Articles

The RUNX1 transcription factor is expressed in serous epithelial ovarian carcinoma and contributes to cell proliferation, migration and invasion

Previously, we have identified the RUNX1 gene as hypomethylated and overexpressed in post-chemotherapy (CT) primary cultures derived from epithelial ovarian cancer (EOC) patients, when compared with primary cultures derived from matched primary (prior to CT) tumors. Here we show that RUNX1 displays a trend of hypomethylation, although not significant, in omental metastases compared with primary EOC tumors. Surprisingly, RUNX1 displayed significantly higher expression not only in metastatic tissue, but also in high-grade primary tumors and even in low malignant potential tumors. The RUNX1 expression levels were almost identical in primary tumors and omental metastases, suggesting that RUNX1 hypomethylation might have a limited impact on its overexpression in advanced (metastatic) stage of the disease. Knockdown of the RUNX1 expression in EOC cells led to sharp decrease of cell proliferation and induced G1 cell cycle arrest. Moreover, RUNX1 suppression significantly inhibited EOC cell migration and invasion. Gene expression profiling and consecutive network and pathway analyses confirmed these findings, as numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, including EOC tumor invasion and metastasis, were found to be downregulated upon RUNX1 suppression, while a number of pro-apoptotic genes and some EOC tumor suppressor genes were induced. Taken together, our data are indicative for a strong oncogenic potential of the RUNX1 gene in EOC progression and suggest that RUNX1 might be a novel EOC therapeutic target. Further studies are needed to more completely elucidate the functional implications of RUNX1 and other members of the RUNX gene family in ovarian tumorigenesis.

Aurora kinase A mediates epithelial ovarian cancer cell migration and adhesion.

Aurora kinase A (AURKA) localizes to centrosomes and mitotic spindles where it mediates mitotic progression and chromosomal stability. Overexpression of AURKA is common in cancer, resulting in acquisition of alternate non-mitotic functions. In the current study, we identified a novel role for AURKA in regulating ovarian cancer cell dissemination and evaluated the efficacy of an AURKA-selective small molecule inhibitor, alisertib (MLN8237), as a single agent and combined with paclitaxel using an orthotopic xenograft model of epithelial ovarian cancer (EOC). Ovarian carcinoma cell lines were used to evaluate the effects of AURKA inhibition and overexpression on migration and adhesion. Pharmacological or RNA interference-mediated inhibition of AURKA significantly reduced ovarian carcinoma cell migration and adhesion and the activation-associated phosphorylation of the cytoskeletal regulatory protein SRC at tyrosine 416 (pSRC(Y416)). Conversely, enforced expression of AURKA resulted in increased migration, adhesion and activation of SRC in cultured cells. In vivo tumor growth and dissemination were inhibited by alisertib treatment as a single agent. Moreover, combination of alisertib with paclitaxel, an agent commonly used in treatment of EOC, resulted in more potent inhibition of tumor growth and dissemination compared with either drug alone. Taken together, these findings support a role for AURKA in EOC dissemination by regulating migration and adhesion. They also point to the potential utility of combining AURKA inhibitors with taxanes as a therapeutic strategy for the treatment of EOC patients.

The role of LPA and YAP signaling in long-term migration of human ovarian cancer cells.

BackgroundThe Hippo-YAP signaling pathway is altered and implicated as oncogenic in many human cancers. However, extracellular signals that regulate the mammalian Hippo pathway have remained elusive until very recently when it was shown that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) ligands including lysophosphatidic acid (LPA) and sphingosine 1-phosphophate (S1P). LPA inhibits Lats kinase activity in HEK293 cells, but the potential involvement of a protein phosphatase was not investigated. The extracellular regulators of YAP dephosphorylation (dpYAP) and nuclear translocation in epithelial ovarian cancer (EOC) are essentially unknown.ResultsWe showed here that LPA dose- and time-dependently induced dpYAP in human EOC cell lines OVCA433, OVCAR5, CAOV3, and Monty-1, accompanied by increased YAP nuclear translocation. YAP was involved in LPA-induced migration and invasion of EOC cells and LPA3 was a major LPA receptor mediating the migratory effect. We demonstrated that G13, but not or to a lesser extent G12, Gi or Gq, was necessary for LPA-induced dpYAP and its nuclear translocation and that RhoA-ROCK, but not RhoB, RhoC, Rac1, cdc42, PI3K, ERK, or AKT, were required for the LPA-dpYAP effect. In contrast to results in HEK293 cells, LPA did not inhibit Mst and Lats kinase in OVCA433 EOC cells. Instead, protein phosphatase 1A (PP1A) acted down-stream of RhoA in LPA-induction of dpYAP. In addition, we identified that amphiregulin (AREG), a down-stream target of YAP which activated EGF receptors (EGFR), mediated an LPA-stimulated and EGFR-dependent long-term (16 hr) cell migration. This process was transcription- and translation-dependent and was distinct from a transcription- and YAP-independent short-term (4 hr) cell migration. EOC tissues had reduced pYAP levels compared to normal and benign ovarian tissues, implying the involvement of dpYAP in EOC pathogenesis, as well as its potential marker and/or target values.ConclusionsA novel LPA-LPA3-G13-RhoA-ROCK-PP1A-dpYAP-AREG-EGFR signaling pathway was linked to LPA-induced migration of EOC cells. Reduced pYAP levels were demonstrated in human EOC tumors as compared to both normal ovarian tissues and benign gynecologic masses. Our findings support that YAP is a potential marker and target for developing novel therapeutic strategies against EOC.

Elevated and secreted phospholipase A₂ activities as new potential therapeutic targets in human epithelial ovarian cancer.

Ascites in epithelial ovarian cancer (EOC) promotes tumor development by mechanisms that are incompletely understood. Lysophosphatidic acid (LPA), a major tumor-promoting factor in EOC ascites, is an enzymatic product of autotaxin (ATX) and phospholipase A(2) (PLA(2))enzymes. The contribution of PLA(2) activities to ovarian tumorigenesis was investigated. The quantitative measurement of PLA(2) activities in ascites and tissues, as well as assay conditions selective for PLA(2) subtypes, were optimized and validated. PLA(2) activities correlated with tumor-promoting activates in cell-based and in vivo assays. High activities consistent with both cytosolic and calcium-independent PLA(2) were found in human EOC ascites for the first time. Elevated PLA(2) and ATX activities were also observed in EOC compared to benign tumors and normal tissues. Cell-free and vesicle-free (S4) human EOC ascites potently promoted proliferation, migration, and invasion of human EOC cells in a PLA(2)-dependent manner. LPA mediated a significant part of the cell-stimulating effects of ascites. S4 ascites stimulated tumorigenesis/metastasis in vivo, and methyl arachidonyl fluorophosphonate was highly effective in inhibiting EOC metastasis in mouse xenograft models. PLA(2) activity was found in conditioned media from both EOC cells and macrophages. Collectively, our work implies that PLA(2) activity is a potential marker and therapeutic target in EOC.

Abstract 4275: PLA2 activities in human epithelial ovarian cancer ascites

Abstract Ascites production is characteristic of late stage human epithelial ovarian cancer (EOC) and it correlates with tumor spread clinically. However, the role of this microenvironment in cancer cells and the mechanisms by which ascites promotes tumor development are not well understood. In recent years, phospholipase A2 (PLA2) enzymes have been identified as cancer therapeutic targets focusing mainly on secreted PLA2s (sPLA2s). In this work, we optimized and validated the quantitative analytic methods to measure PLA2 activities in human EOC ascites and tissues. We found, for the first time, that both cytosolic PLA2 (cPLA2) and calcium-independent PLA2 (iPLA2), but not sPLA2 and autotaxin (ATX), activities (not their expression levels) in human EOC tissues and ascites (the tumor microenvironment) were significantly elevated. In addition, while cell-free (S1) and vesicle-free (S4) human EOC ascites had potent promoting activities in proliferation, migration, and invasion of human EOC cells, the PLA2 activities were involved and account for a significant portion of these activities. Moreover, in vivo studies demonstrated that methyl arachidonyl fluorophosphonate (MAFP, a dual inhibitor of cPLA2 and iPLA2), was highly effective in inhibiting EOC tumorigenesis/metastasis in xenograft models, supporting PLA2 activities as new targets for EOC. LPA, an enzymatic product of ATX and PLA2 enzymes, mediates a significant part of the cellular effects of ascites as evidenced by 1) LPA is produced in cell-free ascites; 2) LPA production and their sensitivities to inhibitors correlate well with the cellular effects in ascites; and 3) the cellular activities were sensitive to LPAR inhibitor and siRNA against LPARs. While cPLA2 and iPLA2 are cytosolic enzymes, our findings indicate that they are associated with cell- and microvesicle-free ascites. In summary, our work implies the potential marker and therapeutic values of PLA2 activity in ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4275. doi:1538-7445.AM2012-4275

Protein Kinase A Activity and Anchoring Are Required for Ovarian Cancer Cell Migration and Invasion

Epithelial ovarian cancer (EOC) is the deadliest of the gynecological malignancies, due in part to its clinically occult metastasis. Therefore, understanding the mechanisms governing EOC dissemination and invasion may provide new targets for antimetastatic therapies or new methods for detection of metastatic disease. The cAMP-dependent protein kinase (PKA) is often dysregulated in EOC. Furthermore, PKA activity and subcellular localization by A-kinase anchoring proteins (AKAPs) are important regulators of cytoskeletal dynamics and cell migration. Thus, we sought to study the role of PKA and AKAP function in both EOC cell migration and invasion. Using the plasma membrane-directed PKA biosensor, pmAKAR3, and an improved migration/invasion assay, we show that PKA is activated at the leading edge of migrating SKOV-3 EOC cells, and that inhibition of PKA activity blocks SKOV-3 cell migration. Furthermore, we show that while the PKA activity within the leading edge of these cells is mediated by anchoring of type-II regulatory PKA subunits (RII), inhibition of anchoring of either RI or RII PKA subunits blocks cell migration. Importantly, we also show – for the first time – that PKA activity is up-regulated at the leading edge of SKOV-3 cells during invasion of a three-dimensional extracellular matrix and, as seen for migration, inhibition of either PKA activity or AKAP-mediated PKA anchoring blocks matrix invasion. These data are the first to demonstrate that the invasion of extracellular matrix by cancer cells elicits activation of PKA within the invasive leading edge and that both PKA activity and anchoring are required for matrix invasion. These observations suggest a role for PKA and AKAP activity in EOC metastasis.

Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

In attempt to discover novel aberrantly hypermethylated genes with putative tumor suppressor function in epithelial ovarian cancer (EOC), we applied expression profiling following pharmacologic inhibition of DNA methylation in EOC cell lines. Among the genes identified, one of particular interest wаs DOK1, or downstream of tyrosine kinase 1, previously recognized as a candidate tumor suppressor gene (TSG) for leukemia and other human malignancies. Using bisulfite sequencing, we determined that a 5′-non-coding DNA region (located at nt −1158 to −850, upstream of the DOK1 translation start codon) was extensively hypermethylated in primary serous EOC tumors compared with normal ovarian specimens; however, this hypermethylation was not associated with DOK1 suppression. On the contrary, DOK1 was found to be strongly overexpressed in serous EOC tumors as compared to normal tissue and importantly, DOK1 overexpression significantly correlated with improved progression-free survival (PFS) values of serous EOC patients. Ectopic modulation of DOK1 expression in EOC cells and consecutive functional analyses pointed toward association of DOK1 expression with increased EOC cell migration and proliferation, and better sensitivity to cisplatin treatment. Gene expression profiling and consecutive network and pathway analyses were also confirmative for DOK1 association with EOC cell migration and proliferation. These analyses were also indicative for DOK1 protective role in EOC tumorigenesis, linked to DOK1-mediated induction of some tumor suppressor factors and its suppression of pro-metastasis genes. Taken together, our findings are suggestive for a possible tumor suppressor role of DOK1 in EOC; however its implication in enhanced EOC cell migration and proliferation restrain us to conclude that DOK1 represents a true TSG in EOC. Further studies are needed to more completely elucidate the functional implications of DOK1 and other members of the DOK gene family in ovarian tumorigenesis.

Combination Therapy of an Inhibitor of Group VIA Phospholipase A 2 with Paclitaxel Is Highly Effective in Blocking Ovarian Cancer Development

We and others have shown that calcium-independent phospholipase A(2) (iPLA(2)) is involved in epithelial ovarian cancer (EOC). Hence, we propose that iPLA(2) is a potential effective and novel target for EOC. We tested this concept and found that bromoenol lactone (BEL), a selective inhibitor of iPLA(2), significantly inhibited EOC metastatic tumor growth in mouse xenograft models using human SKOV3 and HEY ovarian cancer cells. Moreover, the combination of BEL with paclitaxel (PTX), one of the most commonly used therapeutic agents in EOC, almost completely blocked tumor development in the xenograft mouse model. BEL showed no detectable cytotoxic effects in mice. Another iPLA(2) inhibitor, FKGK11, also inhibited tumor development in the xenograft mouse model, supporting that the major target of action was iPLA(2). The additional effects of BEL with PTX in vivo likely stem from their distinct cellular effects. BEL and FKGK11 reduced adhesion, migration, and invasion of EOC cells in vitro; the reduced ability to adhere, migrate, and invade seems to increase the vulnerability of tumor cells to PTX. These results provide an important basis for the development of new treatment modalities for EOC.

Effects of dihydroartiminisin on the adhesion, migration, and invasion of epithelial ovarian cancer cells

To investigate the effects of dihydroartiminisin (DHA) on the adhesion, migration, and invasion ovarian cancer cells. Human ovarian cancer cells of the lines SKOV3 and OVCAR3 were cultured. Suspensions of SKOV3 and OVCAR3 cells were treated with DHA of the concentrations of 0.5, 2.5, 12.5, and 62.5 micromol/L respectively, and then inoculated on the plate coated with Matrigel. MTT method was used to -determine the adhesion rate. Transwell membrane chamber model was used to evaluate the effect of DHA on the migration and invasion of the SKOV3 and OVCAR3 cells. Western blotting and reverse transcriptase polymerase chain reaction were used to detect the effect of DHA on the phosphorylation of focal adhesion kinase (FAK) and on the effect of expression of metal matrix proteinases (MMPs) and their tissue inhibitors (TIMPs) respectively. (1) Compared to the cells without DHA treatment, the cell adhesion ability levels of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA decreased by 76.1% and 57.9% respectively (P < 0.05), while their migration ability levels decreased by 59.3% and 69.7% respectively (P < 0.05). (2) Both SKOV3 and OVCAR3 showed weak invasion ability, and DHA only showed a slight inhibitory effect on the cell invasion of these 2 lines (both P > 0.05). (3) Compared to the cells without DHA treatment, the phosphorylation level of FAK of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA decreased by 42.9% and 44.8% respectively (both P < 0.05). (4) RT-PCR showed mRNA expression of MMP2, TIMP1, and TIMP2, but not mRNA expression of MMP9 in both SKOV3 and OVCAR3 cells. The mRNA expression levels of the SKOV3 and OVCAR3 cells treated with 12.5 micromol/L DHA increased by 1.5 and 2.6 times respectively (both P < 0.05). DHA has inhibitory effects on the adhesion and migration of epithelial ovarian cancer cells, which may be related to its down-regulation of the phosphorylation of FAK in these cells.